The present invention is directed to novel hydrogen phosphonodithioates, and in particular to nucleosides, deoxynucleosides, nucleotides and deoxynucleotides containing novel hydrogen phosphonodithioate groups.
Internucleotide phosphate analogs have recently gained interest as having uses in and of themselves, other than for making recombinant DNA for application in genetic engineering techniques. For example, methanephosphonate linked deoxyoligonucleotides inhibit viral replication in mammalian cells (Smith, C. C., et al., Proc. Natl. Acad. Sci. USA 83, 2787-2791 (1986)) and some phosphorothioates have been shown to have anti-HIV activity (Matsukura, et al., Proc. Natl. Acad. Sci. USA 84, 7706-7710 (1987)). Internucleotide phosphate analog linkages, such as the phosphoramidate linkages, are useful as an attachment for reporter groups, intercalating agents and cross-linking agents. Such linkages also can be resistant to certain nucleases which would otherwise act upon naturally-occurring phosphate internucleotide linkages. Moreover, the modification of the phosphorus-containing internucleotide linkages has been shown to affect gene expression (Marcus-Sekura, et al., Nucl. Acids Res. 15, 5749-5763 (1987)). Therefore, there is a continuing interest in phosphate analogs for all of these utilities, as well as for use in synthesizing sequence-defined oligonucleotides and analogs thereof.
The present invention provides novel and useful hydrogen phosphonodithioates of the formula I: 
in general wherein each R is independently a substituted or unsubstituted hydrocarbyl group, or substituted or unsubstituted hydrocarbyl group containing one or more heteroatoms; Y is O, S, xe2x80x94NHxe2x80x94 or xe2x80x94NRxe2x80x94; and A+ is a cation. By the term hydrocarbyl it is meant any group which contains at least the elements hydrogen and carbon. This term is not intended to be limited to groups which contain only. hydrogen and carbon.
Particularly preferred compounds are those wherein the R group is a nucleoside, nucleotide, deoxynucleoside,.deoxynucleotide or any protected or modified derivative of any of those groups. The most preferred class comprises those compounds wherein Y is oxygen. The nucleosides, nucleotides, deoxynucleosides or deoxynucleotides may be protected at any functional site on the molecule.
It will be appreciated that as used herein the terms xe2x80x9cnucleosidesxe2x80x9d and xe2x80x9cnucleotidesxe2x80x9d will include those moieties which contain not only the known purine and pyrimidine bases, but also bases which have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, and the like.
Modified nucleosides or nucleotides will also include modifications on the sugar moiety, for example, wherein one or more of the hydroxyl groups are replaced with halogen, or functionalized as ethers, etc.
Examples of modified nucleosides or nucleotides which may comprise R in formula I include, but are not limited to:
In general, the compounds according to the present invention are useful in oligonucleotide synthesis to generate analogous internucleotide linkages. The nucleotide (or nucleoside) monomers and oligomers may also themselves have antiviral activity, anticancer utility, or other therapeutic utility, and may also have diagnostic, pesticidal, agricultural, animal health care, or plant health care uses. In particular, the compounds of the invention are useful research reagents for forming internucleotide linkages. A particularly preferred use of the compounds of the present invention is to form internucleotide hydrogen phosphonothioate linkages, which can, in turn, be converted to other phosphothioate linkages.
In general, and as defined in more detail hereinbelow, the compounds according to the present invention include those in which R may be alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, aralkyl, substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, substituted aryl, or substituted aralkyl, including a protected or unprotected nucleoside, nucleotide, deoxynucleoside or deoxynucleotide.
As described in more detail hereinbelow, the compounds according to the present invention may be prepared by treatment of an acidic azole with phosphorus trihalide to form a reactive intermediate which can be treated directly with the compound ROH, RSH, RNH2 or RRNH, wherein R is as described herein. Subsequent treatment with hydrogen sulfide, followed by a base, results in compounds of the present invention. It will be appreciated that 1,2,4-triazole, tetrazoles, or other reactants may be utilized in the synthesis provided they form an intermediate upon reaction with phosphorus trihalide which, in turn, undergoes the desired addition of the ROH, RSH, RNH2 or RRNH utilized in the synthesis.
In a preferred embodiment of the present invention, the compounds of the invention may be made into activated intermediates by conversion of one of the sulfur atoms to a suitable leaving group, under appropriate conditions, such as a thioalkanoate group, 
This activated intermediate is useful for conversion to other useful compounds, such as by reaction with a nucleoside or nucleotide to form hydrogen phosphonothioate ester-linked nucleosides or nucleotides.
The compounds according to the present invention may be made in general in situ by treatment of one equivalent of a phosphorus trihalide, preferably phosphorus trichloride, in a suitable solvent with an excess of a reactive acidic azole, such as 1,1,4-triazole, in 3 to 4 equivalent excess. Then an equivalent amount or less, of a suitable reagent, ROH, RSH, RNH2 or RRNH, wherein R is as defined herein, can be added in the presence of a base. Then treatment with hydrogen sulfide followed by neutralization, such as with trialkyl ammonium bicarbonate, results in compounds according to the present invention, which can be isolated by chromatography, crystallization, distillation, and the like, by conventional procedures.
While generally the synthesis has been described utilizing 1,2,4-triazole, any other suitable acidic azole may be utilized in the synthesis, since the triazole does not appear in the final compound. Other azoles include, but are not limited to, for example, tetrazoles, and the like. Suitable solvents for the in situ reaction will depend, of course, on the solubility of the particular azole utilized in the initial reaction with the phosphorus trihalide, and upon the solubility of the reagent ROH, RSH, RNH2 or RRNH.
Since R may contain one or more functional groups, these groups will be protected during the synthesis of the compounds according to the present invention, particularly if such functional groups are reactive with electrophilic phosphorus species or hydrogen sulfide under the reaction conditions of the synthesis. Conventional blocking groups may be utilized, such as those used for blocking hydroxyl, sulfhydryl, carboxyl and amino groups in chemical synthesis, particularly those groups used for blocking functionalities in peptide synthesis or oligonucleotide synthesis.
In a preferred embodiment of the present invention, R will be a nucleoside, nucleotide, deoxynucleoside or deoxynucleotide, which contains hydroxyl functionalities as well as amine functionalities in the basic side chains. Under the conditions of reactions for the synthesis of compounds according to the present invention described above, typically the 5xe2x80x2 or 3xe2x80x2 hydroxyl group of the ribonucleoside will be protected by a hydroxyl protecting group, preferably the dimethoxytrityl group. The reactive amine groups on the basic side chains may be protected by an amine protecting group, such as an acyl group or amidine. It will be recognized by those of ordinary skill in the art that many other protecting groups may be utilized which will serve to protect the appropriate functionalities under the conditions of the synthesis of the present invention. Some, but not all, protecting groups and the conditions for their use are shown, for example, in Greene, T. W., Protective Groups in Organic Synthesis, Wiley and Sons, N.Y., 1981.
The cation A+ may be any cation, particularly one which serves as a counterion to the neutralizing agent in the final step of the synthesis of compounds of Formula I. In the final step of the above-described synthesis, excess hydrogen sulfide and the thioacidic proton on the phosphonodithioate group are neutralized by addition of a reagent such as bicarbonate. The counterion to the bicarbonate will form the initial A+ moiety in the compound of the present invention. A particularly preferred neutralizing agent for use in the present synthesis is a trialkylammonium bicarbonate whereby the trialkylammonium ion comprises the A+ counterion in the resulting compound. A particularly preferred salt for the neutralization reaction is triethylammonium bicarbonate.
It will be appreciated that the moiety A+ is a cation which results in treatment of the thioacid made in situ by treatment with hydrogen sulfide. In some instances a cation A+ other than the counterion to the acid used for the neutralization reaction may be desired. To prepare a different salt, the original salt product may be dissolved in an organic solvent, such as dichloromethane, then extracted by contact with an aqueous phase containing the desired cation A+. Examples of other cations include, but are not limited to:
1,5-diazabicyclo[4.3.0]non-5-ene ammonium (DBN)
1,4-diazabicyclo[2.2.2]octane ammonium (DABCO)
1,8-diazabicyclo[5.4.0]undec-7-ene ammonium (DBU)
tetrabutylammonium
tributytlammonium
triethylammonium
diisopropylethylammonium
1,8-bis-dimethylaminonaphthalene ammonium
benzyltrimethylammonium
benzyltriethylammonium
A preferred cation is the DBU cation.
The group R of the compounds according to the present invention may be linear or branched alkyl containing 1 to 20 carbon atoms, such as methyl, propyl, isopropyl, n-butyl, isobutyl, t-butyl, isopentyl, neopentyl, n-octyl, n-dodecyl, and the like. The group R may also be a linear or branched alkenyl group containing 1 to 20 carbon atoms such as isopropenyl, 1,4-butadienyl, 3-octenyl, and the like.
The group R may also be cycloalkyl or cycloalkenyl containing 3 to 20 carbon atoms, including, but not limited to, cyclopropyl, cyclopentyl, cyclohexyl, cyclooctyl, cyclopentenyl, cyclohexenyl, and the like.
The group R may be an aryl group containing 6 to 18 carbon atoms such as phenyl, anthracyl, naphthyl, azulyl, phenanthryl, and the like.
The R group may also be an aralkyl group containing about 7 to 60 carbon atoms, including, but not limited to, benzyl, 4-phenylbutyl, and the like.
The alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl or aralkyl groups may further contain one or more heteroatoms such as oxygen, nitrogen, sulfur, silicon, in the form of functionalities including, but not limited to, ethers, thioates, heterocyclics, carboxylic acids, esters, ketones, and the like.
Additionally, the alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, aralkyl groups optionally containing one or more heteroatoms may contain one or more substituents. Typical substituents include hydroxyl, nitro, sulfhydryl, cyano, alkylcarbonyl, alkoxyl, halo, alkylthio, alkylcarboxyl, amino (including primary, secondary, tertiary or quaternary) group. For example, substituents may be selected from the group consisting of xe2x80x94OH, xe2x80x94NO2, xe2x80x94SH, xe2x80x94CN, Oxe2x95x90CRxe2x80x3, xe2x80x94ORxe2x80x2, xe2x80x94X, xe2x80x94SRxe2x80x2 and xe2x80x94CO2Rxe2x80x3 wherein X is halo, Rxe2x80x2 is a removable protecting group or phosphate group; and Rxe2x80x3 is hydrogen or a linear, branched or cycloalkyl containing 1 to 10 carbon atoms, aryl containing 6 to 10 carbon atoms or aralkyl containing 7 to 20 carbon atoms, optionally containing halo or hydroxy substituents.
A preferred class of compounds are those wherein R is of the formula IIA or IIB: 
wherein B is a protected or unprotected nucleoside base; Z is hydrogen, xe2x80x94OH, xe2x80x94OR3 or alkyl containing 1 to 6 carbon atoms, and each R3 is independently a phosphorus-containing group, heteroatom-containing alkyl, haloalkyl or alkyl of 1-10 carbon atoms, protecting group, aryl or substituted aryl wherein the aryl nucleus contains 6-10 carbon atoms, or aralkyl of 7-20 carbon atoms. For example, R3 may be the phosphorus-containing group xe2x80x94CH2xe2x80x94{circle around (P)}xe2x80x94 wherein {circle around (P)} is phosphorus in any of its valence states. The compounds according to the formula IIA and IIB include, but are not limited to, adenosinyl, guanosinyl, cytidinyl, uridinyl, deoxyadenosinyl, deoxyguanosinyl, deoxycytidinyl, thymidinyl, 2xe2x80x2-methyl-deoxyadenosinyl, 2xe2x80x2-methyl-deoxyguanosinyl, 2xe2x80x2-methyl-deoxycytidinyl and 2xe2x80x2-methyl-thymidinyl. It will be appreciated that a unique stereochemistry is not to be inferred by the formulas IIA or IIB. The formulas are intended to be generic not only to the naturally-occurring stereochemistry (including xcex1 and xcex2 anomeric configurations at the 1xe2x80x2-position) but also to any and all combinations of stereochemical configurations of the chiral sites.
It will be realized that blocking groups may be required on one or more functional groups during the synthesis of compounds according to the present invention. These blocking groups, if required, may be removed by procedures recognized in the art, for example by using mild bases, such as ammonium hydroxide, at room temperature or at elevated temperatures. There are numerous blocking groups for different functionalities which are known in the art which can be removed under similar conditions, such as, blocking groups which are commonly used in peptide synthesis. Other blocking groups may be removed in stepwise manner. For example, esters which may be used to block carboxyl functionalities may be removed by treatment with ammonium hydroxide at approximately room temperature (20xc2x0 C.) and N-acyl blocking groups, such as acetyl, benzoyl, and the like, may be removed by warming an ammonium hydroxide-containing solution to about 50xc2x0 C.
Removal of hydroxyl blocking groups such as dimethoxytrityl, trityl and the like may be accomplished by use of any suitable protic acid in a suitable solvent or a Lewis acid such as zinc bromide, aluminum chloride, boron trifluoride, titanium tetrachloride, nitromethane, tetrahydrofuran, or alcohols, under conditions known in the art.
As previously discussed, the compounds according to the present invention may have antiviral or anticancer activity or other therapeutic applications, as well as diagnostic, agricultural, animal health care, or plant health care uses. In a particularly preferred embodiment of the use of compounds according to the present invention, compounds of the formula I may be activated for substitution on the phosphorus atom by treatment with a reagent which converts one of the sulfur atoms to a suitable leaving group, thus activating the phosphorus atom. A particularly preferred reagent is an acylating agent, such as pivaloyl chloride, which acylates one of the sulfur atoms to form the thioalkanoate leaving group 
The phosphorus atom is thereby activated, and in this instance may be converted to other useful compounds, such as by reaction with a nucleoside or nucleotide having a free hydroxyl group to form a phosphothioate ester by displacement of the 
leaving group.
Thus, it is another aspect of the present invention to provide novel compounds of the formula III 
wherein R is as defined hereinabove in connection with the formula I and xe2x80x94SR1 is a leaving group under appropriate conditions of substitution at the phosphorous atom. The group R1 may therefore be an acyl group such as pivaloyl, adamantyl, acetyl, benzoyl, p-methoxybenzoyl, or other suitable leaving group, such as a thioacyl, phosphoryl or thiophosphoryl group. A preferred acylating agent is an amide or acid halide, particularly an acid chloride, such as acetyl chloride. An acylating agent may also be, for example, an N-acyl azole, or any other reagent wherein the group R1 is activated with a suitable leaving group. Other R1 groups include 
wherein Q is NR2, oxygen or sulfur and T is alkyl of 1-10 carbon atoms, aryl or substituted aryl wherein the aryl nucleus contains 6 to 10 carbon atoms, aralkyl of 7-20 carbon atoms, xe2x80x94NR2R2, xe2x80x94SR2, xe2x80x94OR2; wherein each R2 is independently hydrogen, alkyl of 1-10 carbon atoms, aryl or substituted aryl wherein the aryl nucleus contains 6-10 carbon atoms or aralkyl of 7-20 carbon Moms. It is realized that some R1 groups, such as 
form transient compounds which may subsequently undergo intramolecular rearrangements involving the phosphorus nucleus, however, such transient compounds are deemed to be within the scope of the invention.
The activated compound of formula III above will react with another compound, preferably an unprotected hydroxyl group of a nucleoside or deoxynucleoside, in a suitable solvent, thereby forming a phosphothioate ester of the formula IV: 
It will be understood, of course, that under appropriate and well known conditions of substitution, the group R1Sxe2x80x94 must be a preferred leaving group over the substituent RYxe2x80x94 to obtain the desired product. Thus in the embodiment wherein the phosphorus atom is to undergo substitution by displacement of R1Sxe2x80x94, it is preferred that R in formula III not be another acyl group which would compete as a leaving group with R1Sxe2x80x94.
In order to assist in comprehension and understanding of the invention, the following examples are described, but are not intended to limit the scope of the present invention in any manner.